All references disclosed below are hereby incorporated by reference herein.
The recent sequencing of the human genome have led to the identification of a large number of human gene sequences and sequence variation. Current efforts in proteomics are leading to the identification of the entire complement of proteins in a cell or organism. These developments have produced a wide variety of targets for biological inquiry. For example, numerous assays are carried out in microplate wells. One assay may be effected in each well. Assay reaction may be determined by an optically detectable change. The well density presently available in standard 8×12 cm microplates ranges from 96 wells to 1536 well plates. The diameter of the well bottom detected may range from a few millimeters to fractions of a millimeter, depending on the assay and well density.
In investigating genes, cells and proteins a number of biological techniques is employed requiring a diverse range of instrumentation.
One example of biological technique to analyze nucleic acids and proteins has been gel separation. Nucleic acids and proteins may be electrophoretically separated on gel, such as an agarose or acrylamide gel. The gels may be directly imaged on an area array detector using a stain to label bands of differing molecular weight.
Alternatively, the gels may be blotted onto a membrane and the membrane optically analyzed to detect the protein or nucleic acid sequence. Such separations may include localized separated samples having a wide variety of diameters. Optical systems for imaging gels may have a resolution of 200 to 25 microns to detect targets having a dimension of a millimeter or less. 50 micron resolution for gel targets are common. Optical systems imaging blots may have a resolution selected to image targets 2 millimeter or less. Generally a unique optical system is required for the imaging of such targets.
DNA arrays have emerged as a technology for investigating gene expression and variation. Oligonucleotides may be printed on a impermeable substrate such as a glass slide or the bottom of a well of a multiwell plate. Following hybridization with DNA fragments in a sample the spots or the array may be analyzed to detect hybridization of complimentary strands.
The nucleic acids are printed on the substrate at high densities minimizing the amount of reagents and samples required to effect an assay. A nucleic acid spot on array may be 1 to 100 microns in diameter. Presently a number of dedicated array readers are used for the analysis of nucleic acid arrays. One such system is disclosed in U.S. Pat. No. 5,585,639. In this system a focused illumination beam is scanned over an array and imaged onto a linear detector.
Cells are another biological target of interest. Cells have become an increasing interest as targets in ordered cell arrays. In one type of cell array, plasmids from a plasmid library are bonded to a glass slide. Each plasmid includes encoding cDNA regulated by a eucaryotic expression promoter. Using presently available technology for spotting nucleic acids onto a solid substrate, plasmids may be spotted on the slide at very high densities. Cells are deposited on top of the bonded plasmids. The plasmids are chemically transfected into the cells creating an array of spots of living cells. The cells at each spot express the gene present in the plasmid.
A variety of assays may be effected on the cells in a cell array. These assays include immunologic, histo-chemical, and functional assays. The plasmid libraries used to produce cell arrays may include a variety of different genes, or could include variants of the same gene. Cell arrays provide the opportunity to assay living cells, allowing the function and kinetics of proteins to be investigated. Such cell arrays could be used to express and characterize varied types of proteins. Even complex proteins, such as G-protein coupled receptors, ion channels, and membrane transport proteins could be functionally expressed in cell arrays. In one present assay cell arrays expressing variants of the HIV envelope protein are being investigated for structure, structural correlation with an individual's immune response, and functional interaction with receptors.
Cells and cell-sized targets (such as microbeads) are also commonly assayed in non-ordered formats. For example, cells in a liquid suspension may be added to a microplate well. The cells settle to the bottom of the well, forming a disordered two dimensional array. The cells analyzed may be 2 microns to 10 microns in diameter. Again, both cell arrays and their cell analysis systems have used dedicated optical instrumentation for the analysis of these targets. For intra-cellular imaging (e.g. cell nucleus or organelles) a resolution of 0.2 to 1 microns are needed.
A variety of different detection systems for cell imaging are available.
One type of cell analysis systems are laser-based scanners. Confocal laser imaging systems are disclosed in U.S. Pat. Nos. 6,147,798; 5,091,652; and 5,192,980. Volumetric laser cytometers are disclosed in U.S. Pat. No. 5,547,849. Such scanners are highly sensitive and may be optimized to the specific geometry of a substrate, such as a slide. The illumination wavelength is limited to the wavelength specific to a laser. This may limit the choice of dyes or other fluorescent markers that may be used in a sample. Alternatively, multiple lasers could be used increasing the cost and complexity of imaging systems. Another drawback of such systems is the adverse effect on cell viability of laser scanning. The resolution of such systems are limited to a narrow range of magnifications, generally a single magnification level for dedicated targets such as cell arrays. Imaging using a focused laser spot requires pixel by pixel excitation and scanning. The pixels must be combined to form a “virtual” image. A final limitation is photo-bleaching of dyes by the intense laser light.
An alternative system for detection of cells in ordered or non-ordered arrays are microscope based imaging detectors. One such system is disclosed in U.S. Pat. Nos. 5,989,835 and 6,573,039. Such devices are commonly epi-fluorescence microscopes with white light illumination and CCD detection. Multiple illumination and emission filter combinations allow flexible dye choices, and the dyes may be optimized for higher resolution. However, the limited fields of view of these devices prevent the use of large format CCDs with these devices. Microscope objectives were developed for the aperture of the eye (7 mm) rather than for the larger aperture required of modern CCDs which can exceed 25 mm. Such systems use microscope objectives to allow for variable magnification. However, microscope objectives are not well matched to large format commercially available CCDs. Such systems also are not designed to process the large data sets created by multiple images.
FIG. 1 illustrates the primary elements of the prior art Alpha Array 7000™ produced by Alpha Innotech (San Leandro, Calif.). This system is one instrument for detection of array targets. Related optical systems are described in U.S. Pat. No. 6,271,042. This system includes an arc lamp illumination source 2 producing an illumination beam 22. Illumination beam 22 passes through an optical filter 26 on filter wheel 24. Filter 26 allows transmittal of a selected range of wavelengths of light. Light transmitted through filter 26 is focused into opening 28 of bifurcated optical fiber 3. Bifurcated optical fiber 3 transmits the illumination beam to condenser lens 15. Light passing through lens 15 impinges on the sample substrate 30. The sample substrate 30 is held on multi-position slide holder 6 mounted on sample stage 4. Stage 4 is movable along an X and Y axis, with 1 micron precision. Bifurcated optical fiber 3 allows off-axis illumination of sample.
The illumination light 22 is directed onto a sample on sample substrate 30. Targets upon sample substrate 30 such as a spot on an array, are illuminated. The illumination light excites fluorescent dyes producing fluorescent emission. The emitted light is collected by objective lens 13 which collimates the emission of light beam 40. The beam passes through a filter 17 on filter wheel 16. Filter wheel 16 allows rotation of a number of different filters into the path of collected light. A user selects an appropriate emission filter depending on the dye or dyes used in the assay. In this way scattered illumination light would be filtered out from the collected light. Because such filters work best in locations of parallel rays, the filter wheels 16 is located between objective lens 13 and imaging lens 12 at a position of parallel light rays. The collected light beam 40 passes through imaging lens 12 which focuses the image onto area array detector 11.
The optical configuration allows for a 15 micron resolution, which is sufficient to detect individual cells that are 30 to 50 microns in diameter. This resolution is not sufficient to image intracellular components, or paramaterize or classify cells. The system has a 200 micron depth of field. The system also has a 20 millimeter range of focus. This focal range allows use of taller samples or sample containers, such as microplates. The detector used is 1.3 megapixel cooled charge coupled detector (CCD). This detector has 50% quantum efficiency at 400 nanometers and a 18000 electron quantum well.
The use of a cooled CCD for the detector allows for operation of the system using long integration periods. Long integration periods enhance sensitivity by allowing longer detection intervals during which time more collected light is measured. Alternatively shorter integration times may be used for kinetic studies. Such short integration times lower the sensitivity of detection. The individual image captured view of each CCD exposure may be combined using a cross correlation algorithm into a single mosaic image. At pixel resolution of 15 microns a single microscope slide can be read in 15 seconds and imaged in 5 views. This allows kinetic biological signals to be analyzed over minutes or hours.
The 0.13 numerical aperture lens system is designed to image onto the CCD detector. The lens selection is not based on a microscope objective, allowing for a larger aperture of 25 mm. This lens is selected for use at approximately 0.5 magnification to a large format 1.3 megapixel CCD. This lens also provides a sufficiently large working distance to accommodate the full height of a microplate.
While a number of separate imaging systems exist for specific dedicated targets, a need for more versatile imaging systems remains. The large diversity in the size of biological targets requires a system that has a broad range of magnification levels. Given that the target size may range from a 500 micron diameter target on a blot to a 0.5 micron diameter cell organelle the magnification ideally would span three orders of magnitude. Presently no system allows detection of such a range of target sizes. In addition, the optical aberrations of coma, distortion, and lateral color may be a significant limitation to the imaging capabilities of the system.
A number of currently available systems operate using microscope objective and imaging lens. Such lens are poorly adapted to presently available large format CCD array detectors. As such, the high optical magnification of a microscope objective must be de-magnified to fit such detectors. In addition the systems are often limited either by design of the optics or by design of stage to imaging to one specific type of substrate, such as slides or multiwell plates. This further limits the general utility of such systems and often requires dedicated systems for use with each substrate or sample type.
Typically, systems that use the optics of a microscope use on-axis illumination. As used herein, on-axis illumination means that the illumination light passes through the objective lens before striking the target. This is accomplished by use of a dichroic mirror to reflect the wavelengths of the illumination beam and pass wavelengths of the emitted light from the target. This type of illumination can produce an illumination beam that is highly non-uniform, and it can produce internal reflections that limit the sensitivity of the overall detection system. An alternative is to illuminate off-axis. As used herein, off-axis illumination means that the illumination beam does not pass through the objective lens before striking the target. When using off-axis illumination with a variable magnification system, it is most efficient to use optical means to vary the size of the illumination spot so as to match it to the field of view.
There is also a need to rapidly focus the optics on a substrate. In many systems autofocus is performed by moving the objective lens until the sample comes into focus. However, such relocation of the objective lens changes the magnification. Such a change in magnification makes tiling of views into one mosaic image much more difficult.
Cells provide unique challenges for an imaging system. For cell array applications ideally the cells should remain alive. This requires both an optical system in which the illumination light does not adversely affect the cells, and a system that can provide heating and gas exchange to maintain cells in a viable condition.
Multiplexing has enhanced the value of systems by increasing throughput. For example, the detection of a number of different fluorescent dyes at a single location allows a number of different assays to take place at a single location. An optical system which is able to increase multiplexing would save time and allow efficient use of samples and other resources.